Colorimetric assay kit

Truncal blood was collected at 10 AM following decapitation at sacrifice. Serum was allowed to clot on ice for at least 30 min before centrifugation at 10,000 rpm for 10 min at 4°C. Serum was collected and stored at −20°C until further analysis. Biomarkers were analyzed with the following kits: glucose, triglyceride, and cholesterol with Cayman Colorimetric Assay kits (Ann Arbor, MI), leptin, IGF-1, and adiponectin/Acrp30 with R&D DuoSet ELISA Development Systems (Minneapolis, MN) and insulin with Alpco Mouse Ultrasensitive Insulin ELISA (Salem, NH).

The caspase-3 activity was detected using colorimetric assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, the cells were lysed in lysis buffer on ice for 20 min. After centrifugation, supernatants were incubated with the supplied reaction buffer at 37 °C for 4 h for 2 h in the dark. The reactions were measured by changes at the wavelength of 405 nm using an ELISA reader.

T24 cells were seeded at a density of 1×10⁵ cells/well in 6-well plates and treated with bufalin (10 nM), TRAIL (50 ng/ml), a combination, or neither, and incubated for 24 h at 37°C with 5% CO2. Caspase-3, −8 and −9 activity levels were the measured using colorimetric assay kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. Briefly, the cells were lysed in the supplied lysis buffer from the kits. The supernatant liquid was collected and incubated with the supplied reaction buffer, containing DTT and colorimetric tetrapeptides as follows: An Asp-Glu-Val-Asp-p-nitroaniline (pNA) substrate for caspase-3, an Ile-Glu-Thr-Asp-pNA substrate for caspase-8 and a Leu-Glu-His-Asp-pNA substrate for caspase-9, respectively, at 37°C. The extent of reaction was measured as absorbance at 405 nm using an ELISA reader.

Colorimetric assay kits (R&D Systems, Inc., Minneapolis, MN, USA) were used to measure the enzyme activities of caspase-3 and caspase-8 according to the manufacturer's instructions. After 24 h treatment with cisplatin in the presence or absence of bucillamine, the medium was gently removed and discarded, while the cell pellet was lysed by the addition of the lysis buffer. The cell lysates were incubated on ice for 10 min and centrifuged at 10 000 × g for 1 min at 4 °C. The supernatant was then transferred to a new tube to determine caspase activity. Extracts (50 μl) from cells treated with cisplatin in the presence or absence of bucillamine were added to 50 μl of reaction buffer and 5 μl of caspase-3 (DEVD-pNA) or caspase-8 (IETD-pNA) colorimetric substrate. Each mixture was placed in wells of a 96-well plate and incubated for 1 h at 37 °C. Absorbance at 405 nm was measured using a microtiter plate reader (Molecular Devices Co., Sunnyvale, CA, USA). Caspase activity was normalized to micrograms of protein using a Bio-Rad protein assay kit (Bio-Rad Co., Hercules, CA, USA).

The activities of the caspases (caspase-3, -8 and -9) were determined using colorimetric assay kits (R&D Systems, Minneapolis, MN, USA), following the manufacturer's protocol. Briefly, the cells were lysed in the supplied lysis buffer, and equal amounts of proteins were incubated with the supplied reaction buffer, containing dithiothreitol and tetrapeptides as substrates for each caspase, at 37℃ for 2 h in the dark. The reactions were measured by changes in absorbance at 405 nm using an ELISA reader.